Characterization of yeast mutant strains for starter culture in Arabica coffee fermentation.

Arabica coffee is the most popular and best-selling type of coffee. During coffee fermentation, microorganisms are essential for the production of metabolites and volatile compounds that affect coffee flavor quality. This work aimed to study the mutation, selection, and characterization of the Wickerhamomyces anomalus strain YWP1-3 as a starter culture to enhance the flavor quality of Arabica coffee. The results revealed that six mutants could produce relatively high levels of the pectinase enzyme on pectin agar media and exhibited high activity levels, ranging from 332.35 to 415.88 U/ml in mucilage broth. Strains UV22-2, UV22-3, UV41-1 and UV32-1 displayed higher levels of amylase activity than did the wild type. The UV22-2 and UV22-3 mutants exhibited the highest pectin degradation indices of 49.22% and 45.97%, respectively, and displayed significantly enhanced growth rates in nitrogen yeast base media supplemented with various sugars; thus, these mutants were evaluated for their ability to serve as a starter for fermentation of Arabica coffee. The cupping scores of coffees derived from UV22-2 and UV22-3 were 83.5 ± 1.5 and 82.0 ± 2.14, respectively. The volatile compounds in the roasted coffee fermented by UV22-2 were analyzed by GC‒MS, which revealed higher levels of furfuryl alcohol and furfuryl acetate than did the other samples. These findings suggested that UV22-2 could be an influential starter culture for Arabica coffee fermentation.

Microbial metabolism of caffeine and potential applications in bioremediation.

With increasing global consumption of caffeine-rich products, such as coffee, tea, and energy drinks, there is also an increase in urban and processing waste full of residual caffeine with limited disposal options. This waste caffeine has been found to leach into the surrounding environment where it poses a threat to microorganisms, insects, small animals, and entire ecosystems. Growing interest in harnessing this environmental contaminant has led to the discovery of 79 bacterial strains, eight yeast strains, and 32 fungal strains capable of metabolizing caffeine by N-demethylation and/or C-8 oxidation. Recently observed promiscuity of caffeine-degrading enzymes in vivo has opened up the possibility of engineering bacterial strains capable of producing a wide variety of caffeine derivatives from a renewable resource. These engineered strains can be used to reduce the negative environmental impact of leached caffeine-rich waste through bioremediation efforts supplemented by our increasing understanding of new techniques such as cell immobilization. Here, we compile all of the known caffeine-degrading microbial strains, discuss their metabolism and related enzymology, and investigate their potential application in bioremediation.

Japanese sake making using wild yeasts isolated from natural environments.

Saccharomyces cerevisiae is one of the most important microorganisms for the food industry, including Japanese sake, beer, wine, bread, and other products. For sake making, Kyokai sake yeast strains are considered one of the best sake yeast strains because these strains possess fermentation properties that are suitable for the quality of sake required. In recent years, the momentum for the development of unique sake, which is distinct from conventional sake, has grown, and there is now a demand to develop unique sake yeasts that have different sake making properties than Kyokai sake yeast strains. In this minireview, we focus on "wild yeasts," which inhabit natural environments, and introduce basic research on the wild yeasts for sake making, such as their genetic and sake fermentation aspects. Finally, we also discuss the molecular breeding of wild yeast strains for sake fermentation and the possibility for sake making using wild yeasts.

Effect of the cleaning and disinfection methods on the hygienic conditions of fermentation tanks of table olives (Olea europaea L.) Negrinha de Freixo cultivar.

This study aimed to evaluate and identify the microbial community attached to the surfaces of fermenter tanks used in table olive Negrinha de Freixo cultivar processing through molecular analysis and verify if the cleaning/disinfection was done correctly. Four fermentation tanks previously used in table olive processing were sampled at three different inside areas: upper, middle, and lower. Before sampling, four cleaning/disinfection methods were applied to the tanks, including (i) pressurised water; (ii) a disinfectant product used to clean bowls (Vasiloxe); (iii) 10% sodium hydroxide solution (caustic soda liquid); and (iv) a disinfectant product used by the wine industry (Hosbit). For each sample collected, mesophilic aerobic bacteria, yeast and moulds (YMC), lactic acid bacteria (LAB), as well as total coliforms (TC) and Pseudomonas aeruginosa were evaluated. The results showed significant differences between the different cleaning/disinfection methods applied. The fermenter sanitised with only pressurised water showed a greater abundance of microorganisms than the others. Mesophilic aerobic bacteria were the predominant population, with counts ranging between 2.63 and 5.56 log10 CFU/100 cm2, followed by the moulds (3.11-5.03 log10 CFU/100 cm2) and yeasts (2.42-5.12 log10 CFU/100 cm2). High diversity of microbial communities was observed between the different fermenter tanks. The most abundant species belonged to Aureobasidium, Bacillaceae, Cladosporium, and Rhodotorula genera. LAB, TC, and P. aeruginosa were not detected. This study hopes to improve hygienic conditions and increase the quality assurance and safety of the final product.

Natural trait variation across Saccharomycotina species.

Among molecular biologists, the group of fungi called Saccharomycotina is famous for its yeasts. These yeasts in turn are famous for what they have in common-genetic, biochemical, and cell-biological characteristics that serve as models for plants and animals. But behind the apparent homogeneity of Saccharomycotina species lie a wealth of differences. In this review, we discuss traits that vary across the Saccharomycotina subphylum. We describe cases of bright pigmentation; a zoo of cell shapes; metabolic specialties; and species with unique rules of gene regulation. We discuss the genetics of this diversity and why it matters, including insights into basic evolutionary principles with relevance across Eukarya.

Yeast communities related to honeybees: occurrence and distribution in flowers, gut mycobiota, and bee products.

Honeybee (Apis mellifera) is an important agricultural pollinator and a model for sociality. In this study, a deep knowledge on yeast community characterizing the honeybees' environmental was carried out. For this, a total of 93 samples were collected: flowers as food sources, bee gut mycobiota, and bee products (bee pollen, bee bread, propolis), and processed using culture-dependent techniques and a molecular approach for identification. The occurrence of yeast populations was quantitatively similar among flowers, bee gut mycobiota, and bee products. Overall, 27 genera and 51 species were identified. Basidiomycetes genera were predominant in the flowers while the yeast genera detected in all environments were Aureobasidium, Filobasidium, Meyerozyma, and Metschnikowia. Fermenting species belonging to the genera Debaryomyces, Saccharomyces, Starmerella, Pichia, and Lachancea occurred mainly in the gut, while most of the identified species of bee products were not found in the gut mycobiota. Five yeast species, Meyerozyma guilliermondii, Debaryomyces hansenii, Hanseniaspora uvarum, Hanseniaspora guilliermondii, and Starmerella roseus, were present in both summer and winter, thus indicating them as stable components of bee mycobiota. These findings can help understand the yeast community as a component of the bee gut microbiota and its relationship with related environments, since mycobiota characterization was still less unexplored. In addition, the gut microbiota, affecting the nutrition, endocrine signaling, immune function, and pathogen resistance of honeybees, represents a useful tool for its health evaluation and could be a possible source of functional yeasts. KEY POINTS: • The stable yeast populations are represented by M. guilliermondii, D. hansenii, H. uvarum, H. guilliermondii, and S. roseus. • A. pullulans was the most abondance yeast detective in the flowers and honeybee guts. • Aureobasidium, Meyerozyma, Pichia, and Hanseniaspora are the main genera resident in gut tract.

Carbon efficient production of chemicals with yeasts.

Microbial metabolism offers a wide variety of opportunities to produce chemicals from renewable resources. Employing such processes of industrial biotechnology provides valuable means to fight climate change by replacing fossil feedstocks by renewable substrate to reduce or even revert carbon emission. Several yeast species are well suited chassis organisms for this purpose, illustrated by the fact that the still largest microbial production of a chemical, namely bioethanol is based on yeast. Although production of ethanol and some other chemicals is highly efficient, this is not the case for many desired bulk chemicals. One reason for low efficiency is carbon loss, which decreases the product yield and increases the share of total production costs that is taken by substrate costs. Here we discuss the causes for carbon loss in metabolic processes, approaches to avoid carbon loss, as well as opportunities to incorporate carbon from CO2 , based on the electron balance of pathways. These aspects of carbon efficiency are illustrated for the production of succinic acid from a diversity of substrates using different pathways.

Genome-scale metabolic models reveal determinants of phenotypic differences in non-Saccharomyces yeasts.

BACKGROUND: Use of alternative non-Saccharomyces yeasts in wine and beer brewing has gained more attention the recent years. This is both due to the desire to obtain a wider variety of flavours in the product and to reduce the final alcohol content. Given the metabolic differences between the yeast species, we wanted to account for some of the differences by using in silico models. RESULTS: We created and studied genome-scale metabolic models of five different non-Saccharomyces species using an automated processes. These were: Metschnikowia pulcherrima, Lachancea thermotolerans, Hanseniaspora osmophila, Torulaspora delbrueckii and Kluyveromyces lactis. Using the models, we predicted that M. pulcherrima, when compared to the other species, conducts more respiration and thus produces less fermentation products, a finding which agrees with experimental data. Complex I of the electron transport chain was to be present in M. pulcherrima, but absent in the others. The predicted importance of Complex I was diminished when we incorporated constraints on the amount of enzymatic protein, as this shifts the metabolism towards fermentation. CONCLUSIONS: Our results suggest that Complex I in the electron transport chain is a key differentiator between Metschnikowia pulcherrima and the other yeasts considered. Yet, more annotations and experimental data have the potential to improve model quality in order to increase fidelity and confidence in these results. Further experiments should be conducted to confirm the in vivo effect of Complex I in M. pulcherrima and its respiratory metabolism.

Taxogenomic analysis of a novel yeast species isolated from soil, Pichia galeolata sp. nov.

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446T (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

The prevalence of killer yeasts and double-stranded RNAs in the budding yeast Saccharomyces cerevisiae.

Killer toxins are antifungal proteins produced by many species of "killer" yeasts, including the brewer's and baker's yeast Saccharomyces cerevisiae. Screening 1270 strains of S. cerevisiae for killer toxin production found that 50% are killer yeasts, with a higher prevalence of yeasts isolated from human clinical samples and winemaking processes. Since many killer toxins are encoded by satellite double-stranded RNAs (dsRNAs) associated with mycoviruses, S. cerevisiae strains were also assayed for the presence of dsRNAs. This screen identified that 51% of strains contained dsRNAs from the mycovirus families Totiviridae and Partitiviridae, as well as satellite dsRNAs. Killer toxin production was correlated with the presence of satellite dsRNAs but not mycoviruses. However, in most killer yeasts, whole genome analysis identified the killer toxin gene KHS1 as significantly associated with killer toxin production. Most killer yeasts had unique spectrums of antifungal activities compared to canonical killer toxins, and sequence analysis identified mutations that altered their antifungal activities. The prevalence of mycoviruses and killer toxins in S. cerevisiae is important because of their known impact on yeast fitness, with implications for academic research and industrial application of this yeast species.

Yeasts associated with mines on tree leaves in the urban areas / Levaduras asociadas a minas en hojas de árboles en zonas urbanas

Mines on tree leaves and undamaged leaves were studied to investigate yeast complexes in urban areas (Aesculus hippocastanum, miner — Cameraria ohridella; Betula verrucosa, miner — Caloptilia betulicola; Populus nigra, miner — Lithocolletis populifoliella; Quercus robur, miner — Tischeria companella; Salix caprea, miner — Trachys minuta; Syringa vulgaris, miner — Caloptilia syringella; Tilia cordata, miner — Phyllonorycter issikii; Ulmus laevis, miner — Carpatolechia fugitivella). The abundance and taxonomic structure of yeasts were studied using a surface plating method on solid media (GPY agar). Identification of yeast species was based on the ITS rDNA nucleotide sequence. The average abundance of yeasts during the first stages of mine formation in the internal tissues of leaves was 103 cfu/g. After 23–25 days, during the last stage of larval metamorphosis before mine destruction, the abundance of yeasts in the mines increased by two orders of magnitude to 105 cfu/g. No significant differences were observed in the abundance of yeasts in mines formed by different insects on different trees. A total of twelve yeast species were observed. The fast-growing ascomycetous yeasts Hanseniaspora uvarum and H. occidentalis dominated the mines. On undamaged leaves, the basidiomycetous yeasts Papiliotrema flavescens and Rhodotorula mucilaginosa, typical in the phyllosphere, dominated. The opportunistic yeast Candida parapsilosis was detected in the yeast complexes of all mines examined and was not found on the surface of leaves. Comparison of the relative abundance of yeast species between the studied mines and undamaged leaves using principal component analysis showed that all studied yeast communities in the mines were significantly different from the epiphytic yeast complexes of the undamaged leaves. Thus, miners in urban environments provoke the formation of short-lived endophytic yeast complexes with high abundance of Hanseniaspora. For leaf miners, the yeasts serve primarily as a food source for insect larvae rich in vitamins and amino acids. The adult leaf miners, in turn, contribute to the reproduction of the yeasts and create favorable conditions for their development.(AU)

CAR1 as a new selective marker for genetic engineering of wine yeasts.

A common problem in engineering industrial yeasts, and wine yeasts in particular, is the lack or scarcity of selective markers for introducing desired genetic changes. Almost all such markers, which are usually auxotrophic mutations, would reduce the growth characteristics of yeast strains. However, a potentially useful marker could be the CAR1 gene encoding arginase, the deletion of which reduces the accumulation of the carcinogen ethyl carbamate in wine, making such a deletion beneficial for wine production and maintainable in wine yeast strains. Here we demonstrate the use of the CAR1 gene as a selective marker. First, we observe that complete deletion of CAR1 in a triploid wine strain of Saccharomyces cerevisiae causes strong growth inhibition on a medium containing arginine as the only nitrogen source. Then, we show that strains with CAR1 deletion can be reliably transformed using CAR1 as a plasmid marker. Thus, the CAR1 gene can be used as a convenient selective marker in genetic engineering of wine yeasts, in particular using CRISPR/Cas9 technology.

Dung beetle-associated yeasts display multiple stress tolerance: a desirable trait of potential industrial strains.

BACKGROUND: Stress-tolerant yeasts are highly desirable for cost-effective bioprocessing. Several strategies have been documented to develop robust yeasts, such as genetic and metabolic engineering, artificial selection, and natural selection strategies, among others. However, the significant drawbacks of such techniques have motivated the exploration of naturally occurring stress-tolerant yeasts. We previously explored the biodiversity of non-conventional dung beetle-associated yeasts from extremophilic and pristine environments in Botswana (Nwaefuna AE et.al., Yeast, 2023). Here, we assessed their tolerance to industrially relevant stressors individually, such as elevated concentrations of osmolytes, organic acids, ethanol, and oxidizing agents, as well as elevated temperatures. RESULTS: Our findings suggest that these dung beetle-associated yeasts tolerate various stresses comparable to those of the robust bioethanol yeast strain, Saccharomyces cerevisiae (Ethanol Red™). Fifty-six percent of the yeast isolates were tolerant of temperatures up to 42 °C, 12.4% of them could tolerate ethanol concentrations up to 9% (v/v), 43.2% of them were tolerant to formic acid concentrations up to 20 mM, 22.7% were tolerant to acetic acid concentrations up to 45 mM, 34.0% of them could tolerate hydrogen peroxide up to 7 mM, and 44.3% of the yeasts could tolerate osmotic stress up to 1.5 M. CONCLUSION: The ability to tolerate multiple stresses is a desirable trait in the selection of novel production strains for diverse biotechnological applications, such as bioethanol production. Our study shows that the exploration of natural diversity in the search for stress-tolerant yeasts is an appealing approach for the development of robust yeasts.

Engineered yeasts and lignocellulosic biomaterials: shaping a new dimension for biorefinery and global bioeconomy.

The next milestone of synthetic biology research relies on the development of customized microbes for specific industrial purposes. Metabolic pathways of an organism, for example, depict its chemical repertoire and its genetic makeup. If genes controlling such pathways can be identified, scientists can decide to enhance or rewrite them for different purposes depending on the organism and the desired metabolites. The lignocellulosic biorefinery has achieved good progress over the past few years with potential impact on global bioeconomy. This principle aims to produce different bio-based products like biochemical(s) or biofuel(s) from plant biomass under microbial actions. Meanwhile, yeasts have proven very useful for different biotechnological applications. Hence, their potentials in genetic/metabolic engineering can be fully explored for lignocellulosic biorefineries. For instance, the secretion of enzymes above the natural limit (aided by genetic engineering) would speed-up the down-line processes in lignocellulosic biorefineries and the cost. Thus, the next milestone would greatly require the development of synthetic yeasts with much more efficient metabolic capacities to achieve basic requirements for particular biorefinery. This review gave comprehensive overview of lignocellulosic biomaterials and their importance in bioeconomy. Many researchers have demonstrated the engineering of several ligninolytic enzymes in heterologous yeast hosts. However, there are still many factors needing to be well understood like the secretion time, titter value, thermal stability, pH tolerance, and reactivity of the recombinant enzymes. Here, we give a detailed account of the potentials of engineered yeasts being discussed, as well as the constraints associated with their development and applications.

Metabolic pathways of an organism depict its chemical repertoire and its genetic makeup.Autonomous synthetic microbes can be developed for lignocellulose biorefinery (LCB).LCBs can be harnessed with synthetic microbes to boost global bioeconomy.Yeasts can be engineered to enhance downstream process of LCB.

Biotransformation of ricinoleic acid to γ-decalactone using novel yeast strains and process optimization by applying Taguchi model.

Flavour molecules are generated now-a-days through microbial fermentation on a commercial scale. γ-Decalactone (GDL) is an important molecule due to its long-lasting flavouring impact as buttery, coconut and peach-type. In the current study, 33 microorganisms were isolated from different fruit sources, and their screening for target GDL production was performed. Using DNA sequencing, two potential strains yielding good amounts of GDL were identified from pineapple and strawberry fruits. The identified strains were Metschnikowia vanudenii (OP954735) and Candida parapsilosis (OP954733), and further optimized by Taguchi method. The effectiveness of lactone production is influenced by the rate of microbial growth under various operating conditions. The factors such as substrate concentration, pH, temperature, cell density and rotation (rpm) with 3 levels were applied for the GDL production using M. vanudenii (OP954735) and C. parapsilosis (OP954733) strains. The results revealed that the highest molar conversion of GDL was 24.69% (115.7 mg/g quantitative yield) and 52.69% (272.0 mg/g quantitative yield) at the optimal conditions using SB-62 and PA-19 strains, respectively. The two novel strains are reported for the first time for production of γ-decalactone and overall, this study opens up the possibility of using Taguchi design for large scale up process development for producing food flavours utilising environmentally friendly natural strains.

Metabolic engineering of oleaginous yeast in the lipogenic phase enhances production of nervonic acid.

Insufficient biosynthesis efficiency during the lipogenic phase can be a major obstacle to engineering oleaginous yeasts to overproduce very long-chain fatty acids (VLCFAs). Taking nervonic acid (NA, C24:1) as an example, we overcame the bottleneck to overproduce NA in an engineered Rhodosporidium toruloides by improving the biosynthesis of VLCFAs during the lipogenic phase. First, evaluating the catalytic preferences of three plant-derived ketoacyl-CoA synthases (KCSs) rationally guided reconstructing an efficient NA biosynthetic pathway in R. toruloides. More importantly, a genome-wide transcriptional analysis endowed clues to strengthen the fatty acid elongation (FAE) module and identify/use lipogenic phase-activated promoter, collectively addressing the stagnation of NA accumulation during the lipogenic phase. The best-designed strain exhibited a high NA content (as the major component in total fatty acid [TFA], 46.3%) and produced a titer of 44.2 g/L in a 5 L bioreactor. The strategy developed here provides an engineering framework to establish the microbial process of producing valuable VLCFAs in oleaginous yeasts.

Novel Metschnikowia yeasts from the gut of green lacewing in Japan.

Family Chrysopidae is known to harbor specific gut yeasts. However, no studies have been conducted outside of a limited number of these green lacewing species, and the diversity of yeasts in the family as a whole is not known. Therefore, we collected 58 Chrysopidae adults (9 species, 6 genera, 2 subfamilies) in Japan and isolated yeasts from all individuals. The results showed for the first time that not only subfamily Chrysopinae but also subfamily Apochrysinae have gut yeasts. We obtained 58 yeast isolates (one from each host individual), all of which were of the genus Metschnikowia. 28S rDNA- and ITS-based phylogenetic analysis showed that the isolates were divided into three clades, designated clade I, II, and III. Clade I contains two previously described Chrysopidae gut yeasts (M. picachoensis and M. pimensis) as well as a one of our new species named M. shishimaru. Clade II is a new clade, with at least two new species named M. kenjo and M. seizan. Clade III contains the previously described species M. noctiluminum, a Chrysopidae gut yeast, and one of our isolate (We have not described it as new species). However, the phylogenetic relationship between our isolate and M. noctiluminum was unclear. These results indicate that the Japanese Chrysopidae gut yeasts consist mainly of three undescribed species and that they are more unique than those found in previous surveys. The results of this study indicate that Chrysopidae gut yeasts are more diverse than previously thought and should be investigated in various geographical regions in the future.

Candida massiliensis sp. nov. Isolated from a Clinical Sample.

The majority of Candida species are known as non-pathogenic yeasts and rarely involved in human diseases. However, recently case reports of human infections caused by non-albicans Candida species have increased, mostly in immunocompromised hosts. Our study aimed to describe and characterize as thoroughly as possible, a new species of the Metschnikowia clade, named here Candida massiliensis (PMML0037), isolated from a clinical sample of human sputum. We targeted four discriminant genetic regions: "Internal Transcribed Spacers" of rRNA, D1/D2 domains (28S large subunit rRNA) and part of the genes encoding Translation Elongation Factor 1-α and ß-tubulin2. The genetic data were compared to morphological characters, from scanning electron microscopy (TM 4000 Plus, SU5000), physiological, including the results of oxidation and assimilation tests of different carbon sources by the Biolog system, and chemical mapping by Energy-Dispersive X-ray Spectroscopy. Lastly, the in vitro antifungal susceptibility profile was performed using the E-test™ exponential gradient method. The multilocus analysis supported the genetic position of Candida massiliensis (PMML0037) as a new species of the Metschnikowia clade, and the phenotypic analysis highlighted its unique morphological and chemical profile when compared to the other Candida/Metschnikowia species included in the study.